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Ethanol from Wood. Cellulase Enzyme Production

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Ethanol from Wood. Cellulase Enzyme Production HECSVED OCT 0 6 2000 esTi ETHANOL FROM WOOD CELLULASE ENZYME PRODUCTION ZSOLT SZENGYEL Department of Chemical Engineering 1 Lund University • Sweden Akademisk avhandling for avlaggande av teknologie doktorexamen vid Tekniska fakulteten vid Lunds universitet. Avhandlingen kommer art forsvaras pa engelska vid en ofFentlig disputation pa Kemicentrum, Getingevagen 60, Lund, horsal C, onsdagen den 29 mars 2000 kl. 10.15. Fakultetsopponent ar Prof. Walter Steiner, Inst, fur Biotechnologie, AG Enzymtechnologie, Graz, Austria. Organization Document name Dept, of Chemical Engineering 1, Doctoral Dissertation Lund University Date of issue March, 2000 CODEN LUTKDH/(TKKA-1001)/(l-53)/(2000) Authors) Sponsoring organization: Zsolt Szengyel NUTEK, STEM Title and subtitle Ethanol from wood Cellulase enzyme production Abstract Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated Key words cellulases, production, hardwood, softwood, solid, liquid, carbon source, pH, inhibition, volatile, non-volatile, furfural, acetic acid, FPA measurement Supplementary bibliographical information Language English ISSN and key title 1100-2778 ISBN 91-7874-039-8 Recipients note Number of pages 121 Price Security classification Distribution by (name and address) Dept. ofChem. Eng. 1, Lund University P.O. Box 124, SE-221 00 Lund, Sweden I, the undersigned, being the copyright owner of the abstract of the above-mentioneddissertation, hereby grant to all reference sources permissions to publish and disseminate the abstract of the above-mentioned dissertation. Signature. Date March 01. 2000 11 DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. Errata to OSTI ETHANOL FROM WOOD CELLULASE ENZYME PRODUCTION Zsolt Szengyel, Ph.D. thesis, 2000 Department of Chemical Engineering 1 Lund University, Lund, Sweden page line is should be 9 -8 hydrophobic hydrophilic 10 +12 monopolysaccharide homopolysaccharide 10 +13 glucoside giucosidic 14 Figure 2.2 Hydrolizate Hydrolyzate 16 +3 SPW-S SPS-W 18 Figure 3.1 - "B-Glucosidase" above last arrow 29 Figure 4.2 caption willow (♦) willow (A) 1 -'( Ai •I If we knew what we were doing, it wouldn’t be called research, would it? - Albert Einstein (1879-1955) . i ACKNOWLEDGMENTS I think that this part of the thesis is as difficult to write, and requires as much carefulness, as the rest of the thesis. It is always hard to look back trying to remember all of those kind individuals, who have helped me during the past four and a half years. I would like to say ‘thank you’ to all of them. First of all, I would like to express my sincere gratitude to Prof. Guido Zacchi for giving me the possibility to do my research in Sweden. Thanks for trusting me and my capabilities. Thanks for forming my knowledge, and for bringing the engineering way of thinking a little bit closer to me. Thanks for the fruitful discussions, which were so important to me. Dr. Mats Galbe, with whom I shared a room for two years. Thanks for supplying me with tons of information and having time for discussing the ‘don’t-bother-the-boss-with- this-simple ’ problems. It was great fun tinkering with the broken-down-HPLC, swearing silently. We have spent great times on the Internet laughing about the Hungarian language peculiar phrases. Hope youhave learnt a couple of those! Kerstin (now Dr. Stenberg), and Charlotte for introducing me into the ‘secret world’ of steam pretreatment. I will always remember the ‘small’ ethanol meetings, where we talked a lot about the activity going on in the lab. I owe you a great amount of gratitude for widening my knowledge in the field of ethanol making. A great deal of appreciation goes to Hans-Olof and Leif, for being always around, getting broken down equipment started when I thought nothing could help anymore. I wish to express my thanks to Lill, our wonderful secretary, for giving always maximum assistance in administration matters. I am also grateful to my former supervisor, Dr. Kati Reczey. I think it all started in your lab so many years ago, that I cannot even remember when. Thanks for being my supervisor then, starting my scientific life, and managing my research, even when I thought it was not necessary at all. Koszonettel tartozom sziileimnek, hogy mindvegig bfztak bennem es ha sziikseg volt ra kemeny kezzel, neha ‘ostorral ’ gydztek meg arrol, hogy mi az elet helyes utja, ami persze nem mindig volt olyan konnyu. Gondoskodasotokert, kitartasotokert mindig halas maradok! Koszi Anya ds Apa! V LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to by their Roman numerals in the text. I. Reczey, K., Szengyel, Zs., Eklund, R. and Zacchi, G. (1996) Cellulase production by T. reesei. Bioresour. Technol. 57, 25-30 II. Szengyel, Zs., Zacchi, G. and Reczey, K. (1997) Cellulase production based on hemicellulose hydrolyzate from steam pretreated willow. Appl. Biochem. Biotechnol. 63-65, 351-362 III. Szengyel, Zs. and Zacchi, G. (1999) The effect of acetic acid and furfural on the cellulase production of Trichoderma reesei RUT C30. (Submitted) IV. Szengyel, Zs. and Zacchi, G. (1999) Production of cellulases by Trichoderma reesei RUT C30 using various carbon sources based on steam pretreated spruce. (Submitted) V. Szengyel, Zs., Zacchi, G., Varga, A. and Reczey, K. (2000) Cellulase production of Trichoderma reesei Rut C30 using steam pretreated spruce. The hydrolytic potential of cellulases on different substrates. Appl. Biochem. Biotechnol. (Accepted) Other related papers by the same author: 1. Palmqvist, E., Hahn-Hagerdal, B., Galbe, M., Larsson, M., Stenberg, K., Szengyel, Zs., Tengborg, C., and Zacchi, G. (1996) Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics. Bioresour. Technol. 58, 171-179 2. Palmqvist, E., Hahn-Hagerdal, B., Szengyel, Zs., Zacchi, G. and Reczey, K. (1997) Simultaneous detoxification and enzyme production of hemicellulose hydrolyzates obtained after steam pretreatment. Enzyme Microbiol. Technol. 20, 286-293 3. Reczey, K., Brumbauer, A., Bollok, M., Szengyel, Zs. and Zacchi, G. (1998) Use of hemicellulose hydrolyzate for fi-glucosidase fermentation. Appl. Biochem. Biotechnol. 70-72, 225-235 VI CONTENTS 1. INTRODUCTION 3 1.1 CONVERSION OF LIGNOCELLULOSIC BIOMASS TO ETHANOL 3 1.2 THE ENZYMATIC PROCESS 6 1.3 WHY ETHANOL? 6 1.4 OUTLINE OF THE THESIS 6 2. LIGNOCELLULOSIC BIOMASS 9 2.1 STRUCTURE 9 2.2 CHEMICAL COMPOSITION 9 2.2.1 Cellulose 10 2.2.2 Hemicellulose 11 2.2.3 Lignin 11 2.2.4 Other Components, Extractives 11 2.2.5 Characteristic Differences Between Softwood and Hardwood 11 2.3 PRETREATMENT 12 3. CELLULASES 17 3.1 FUNGAL CELLULASES 17 3.2 MEASUREMENT OF CELLULASE ACTIVITY 18 4. CELLULASE PRODUCTION BY TRICHODERMA 23 4.1 STRAIN DEVELOPMENT 23 4.2 CARBON SOURCE 24 4.2.1 Effect of Delignification of Steam Pretreated Willow 25 4.2.2 Effect of Carbon Source Concentration 25 4.2.3 Substitution of Solid Carbon Source with Liquid Carbon Source 26 4.2.4 Comparison of Steam Pretreated Hardwood and Softwood 29 4.3 EFFECT OF CULTIVATION pH 30 4.4 EFFECT OF VOLATILE AND NON-VOLATILE INHIBITORS 31 4.5 ACETIC ACID AND FURFURAL INHIBITION 34 5. HYDROLYSIS OF LIGNOCELLULOSIC SUBSTRATES 41 6. CONCLUSIONS 45 7. REFERENCES 47 ETHANOL FROM WOOD CELLULASE ENZYME PRODUCTION ETHANOL FROM WOOD CELLULASE ENZYME PRODUCTION Copyright © 2000 by Zsolt Szengyel All rights reserved Printed in Sweden by KFS AB, Lund, 2000 DEPARTMENT OF CHEMICAL ENGINEERING 1 LUND UNIVERSITY P.O. BOX 124 SE-221 00 LUND, SWEDEN ISSN 1100-2778 ISRN LUTKDH/(TKKA-1001)/(l-53)/(2000) ISBN 91-7874-039-8 1. INTRODUCTION The conversion of biomass to liquid fuels, such as ethanol, has been the focus of much interest during the 20th century. The process of making alcohol from cellulose, in principle, is relatively simple: after hydrolysis of cellulose to glucose and a subsequent fermentation, the ethanol can be refined by distillation. Possessing an economically feasible technology would reduce crude oil dependency. Ethanol was first made from cellulose in large-scale production during World Wars I and II using acid hydrolysis, and was later refined during the 1940s. However, due to low yields in the hydrolysis step and corrosion problems caused by the acid catalyst used, the process was not competitive with petroleum-based processes [1], During the oil crisis of the 1970s alternative fuels came into the picture again and were considered economically feasible.
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